JPH02109982A - Gene fragment, probe and detection of chromosomal aberration - Google Patents

Abstract

PURPOSE:To detect aberration of chromogene by forming a gene fraction comprising a hybridizing nucleic acid molecule at least part of a specific site in a primary intron of BCR gene on a human 22nd chromosome. CONSTITUTION:A probe is formed from a gene fragment which comprises a nucleic acid molecule to hybridize at least part from a position going up to the upper steam side by 1Kb from the third EroRI scission site at the 3' side in a primary intron of BCR gene on a human 22nd chromosome to the second Hind III scission site from the 3' side and labeled with a detectable substance. Genome DNA of specimen is scissored with a restriction enzyme, Southern hybridization is carried out by using the probe, then a band is detected followed by chromosomal aberration detection.

Description

Detailed Description of the Invention a. Industrial Application Field The present invention provides gene fragments and probes useful for detecting chromosomal gene abnormalities, specifically chromosomal gene abnormalities based on chromosomal gene translocations, and the like. This relates to the detection method used.

b. Prior Art Chromosomal gene abnormalities, particularly chromosomal gene translocations, are known to be associated with human cancer. It is known that in chronic myeloid leukemia (CML) there is a Philadelphia chromosome that results from a translocation of chromosomes 9 and 22 (Rowley, J. D., Nat.
ure 243.290 (1973)).

In normal cells, when the tumor gene c-abl translocates with chromosome 9, c-ab! It breaks near the gene, and this is the 22nd 8CR (breakpoint cluster).
The bcr-abl gene is ligated to the cut region in a region called the er region, which synthesizes mRNA different from normal, and this mRNA produces a protein with tyrosine kinase activity. In other words, it is thought that chromosomal translocation activates myogenic genes, resulting in CML (de Klein, A. et al., Na.
ture, 300.765 (1982): Heis
terkam, N., et al., Nature
, 306°239 (19B3): and Groff
en, J. , et al., Ce1l.

36, 93 (1984)).

Methods for detecting chromosomal abnormalities in CML have also been developed (for example, U.S. Patent No. 571
, No. 911 (filed January 18, 1984) and U.S. Pat.

Furthermore, C derived from the bcr-ab l gene mentioned above
[)NA has also been isolated, and detailed studies on chromosomal translocations have been conducted (Hariharan, 1.
and Adams.

J, H,, EH11J,, 6.115-119 (1
987)).

Recently, it has been reported that chromosomes 9 and 22 are translocated in acute lymphoblastic leukemia (ALL), and the location of the translocation is different from that in CML (Herman S, A. , et al.

Ce1l 51.33-40 (1987) and Rub
in, C,H,, etal,, Proc, Nat
l, Acad, Sci, USA 85.2795
-2799 (1988)).

C6 Structure of the Invention The present invention provides gene fragments, probes, and methods for efficiently and easily detecting the translocation between chromosome 9 and chromosome 22 in ALL.
Facilitates genetic diagnosis of cases where translocation occurs in the first intron of Butsuko. Therefore, the present invention has the following features: 1. From a position 1 kb 5° upstream of the third EcoRI cleavage site from the 3' side in the first intron of the BCR gene on human chromosome 22, A gene fragment consisting of a nucleic acid molecule that hybridizes to at least a portion of the region up to the second old ndll cleavage site.

2. From the position 1 kb upstream of the third EcoRI cleavage site from the 3° side of the first inn 1 to ron of the BCR gene on human chromosome 22, A gene fragment consisting of an RNA molecule that hybridizes to at least a portion up to the old ndll cleavage site.

3. In the first intron of the BCR gene on human chromosome 22, from the position 1 kb 5° upstream from the third Eco RI cleavage site from the 3° side, to the second intron from the 3' side. D to HindIII cleavage site
Ba-HI-BamHI in the NA sequence.

Pvu I[-Pvu If, or Xho I-Xh
o A gene fragment consisting of a nucleic acid molecule that hybridizes to the cut site 500 bases upstream from I.

4. In the first intron of the BCR gene on human chromosome 22, from the position 1 kb 5' upstream from the third Eco RI cleavage site from the 3° side, the second intron from the 3" side. D up to the old ndI[I cleavage site
Ba-HI-BamHI, Pv in the NA sequence
A gene fragment consisting of an RNA molecule that hybridizes to the cut site 500 bases upstream from u■-pvuI or Xho I-Xho I.

5. The above-mentioned pipe redization is the above-mentioned Bam HI
-Bant-II, Pvu fl -Pvu
The gene fragment according to item 3 or 4 above, which corresponds substantially completely to the DNA sequence of the cleavage site 500 bases upstream from fl or Xho I.

6. A probe consisting of the gene fragment of any one of items 1 to 5 above that is labeled with a detectable substance, and 7. After cutting the genomic DNA of the subject with a restriction enzyme, the probe is prepared as described in item 6 above. A method for detecting chromosomal abnormalities, which comprises performing Southern hybridization using a probe and then detecting a band.

It is.

The present inventors conducted research on the chromosomal translocation that occurs in the first intron of the BCR gene on human chromosome 22 in ALL, and found that the Eco RI cleavage site is located at the third position from the 3' side of the first intron. 5゛ upstream from 1
We succeeded in isolating a new NA fragment of about 7.5 kb from the 3" side to the second old nd[[ cut site, and used this partial DNA fragment as a probe. When analyzing the chromosomal DNA of ALL patients, they discovered that a chromosomal translocation had occurred in this region.

That is, before and after chromosomal translocation, the restriction enzyme map in the vicinity of the point where the translocation occurred changes, and the size of the DNA fragments produced by each restriction enzyme changes compared to the normal state. Therefore, patient genomic DNA and normal person genomic DNA
After cutting A with an appropriate restriction enzyme and performing Southern hybridization using DNA near the translocation site as a probe, a band of a size not seen in normal humans was observed, indicating that translocation had occurred. Recognize.

These differences in DNA fragments between normal people and ALL patients are detected using typical restriction enzymes EC0RI and HindIII.

A representative example using 8amHI and ag+n is shown in Table 1 below. .

Table 1 Examples of differences in gene fragments The above examples are examples of DNA fragments obtained when specific restriction enzymes are used, but even if different restriction enzymes are used, the lengths of the DNA fragments themselves may differ. Also, DNA fragments with different lengths occur between normal people and ALL patients, which can be detected by using the probe of the present invention.

It should be noted that the reason there is a difference between normal individuals X, Y, and Z is thought to be due to polmorphism in intron ①, but this does not affect the identification of ALL patients.

Furthermore, as shown in the Examples below, this ALL-2 patient was completely cured by radiation therapy and the like after the onset of the disease. As a result of similar analysis using EC0RI as a restriction enzyme using the genomic DNA of a former patient after a complete recovery, only one band b was observed at approximately 8.5. This shows that leukocytes in which chromosome translocation occurred have been completely removed from the former patient after complete recovery, and this also shows that the detection method of the present invention is accurate.

Such a probe in the present invention includes the above-mentioned BCRm
If it hybridizes with the newly discovered DNA fragment of about 7.5 to b in the first intron of the gene, DNA, RNA
A. It may be a mixture of DNA and RNA. Also, its length
may have various lengths depending on the hybridization conditions, but should be at least 15 bases or more, preferably 20 bases or more.
Various types can be considered as it only needs to be a base or more. Furthermore, as a method for labeling DNA or RNA used as a probe, radioactive isotopes, fluorescent substances, phosphorescent substances, enzymes, biotin, avidin, etc. can be used.

Also, using the probe described here as a brimer,
It can also be detected by a method that applies a gene amplification reaction using polymerase.

EXAMPLE 1 Isolation of BCR gene 1 Preparation of diagnostic probe 2 Preparation of diagnostic RN and A probes and Southern hybridization will be described below with reference to Examples.

Example 1 Isolation of BCRM gene Hariharan et al.
and Adams J, H, EHBOJ, 6.1
15-119 (1987)) corresponding to the 70 bp of the second exon of the BCRM gene, and the two complementary nucleotide sequences thereto.
Seed DNA was synthesized using a DNA synthesizer (manufactured by Applied Biosystems). After purification, these two synthetic NAs were each treated with 10 mHTr at a degree of 50/l/d.
is-C! 2 (pH 7.6) and 1 mM EDTA, heated in a 70'CF water bath for 30 minutes, and then naturally cooled to room temperature to anneal the two synthetic NAs. This double-stranded DNA was labeled with 32P by nick translation using α[32P]-dCTP,
Approximately 500,000 plaques of a human placenta-derived chromosomal gene library prepared via an EcoRI linker were screened. As a result, a clone of λBCR64 was obtained (
(See attached Figure 1 (4)). Furthermore, the above library was screened (gene walking) using a DNA fragment 5° upstream of the λBCR64 clone as a probe, and a clone containing the 5° upstream side of λBCR64, λB
I took 1q of CR65. A restriction enzyme map was created for each clone using various restriction enzymes.

The results are shown in attached Figure 1 (2). From now on, λB
The approximately 7.5 Kb gene from the 5° end of CR65 to the ndIII cleavage site of the first restriction enzyme has not been previously reported and was found to be a newly obtained portion.

Example 2 Diagnostic probe production The insert DNA of λBCR65 was digested with restriction enzyme Ec.

After digestion with Ri and
e^). λBCR65 was digested with the restriction enzyme Pvu ■, and the resulting DNA fragment of approximately 0.5 Kb was isolated and designated as probe [3 (probe B). Also,
λBCR65 was digested with the restriction enzyme BamHI, and the resulting DNA fragment of approximately 1.3 b was isolated and designated as probe C (probe c).

To create the above nuclear probe, α[32
P] - labeled by nick translation using dCTP.

Example 3 Diagnostic RNA probe production RiboprobeGiemini system (RiboprobeG)
The vector pSP72 of emini System; manufactured by Promega was cut with the restriction enzyme BamHI, and the vector pSP72 was digested with bovine alkaline phosphatase (Bovine Alk
Aline Phospha-tase) treatment was performed. In addition to this, probe C (probe C prepared in Example 2)
beC) and ligated with DNA Ligase to form 1)S
It was designated as P72-PRC. Using this plasmid, Melton's method (Melton, D.A. etat., N.
uc, Ac1d Res, t2.7035-705
6 (1984))-C probe C DNA
An RNA probe corresponding to the base sequence was synthesized. First p
SP72-PRC was cut with restriction enzyme Sma I and AT
P.GTP.

UTP and α[32P]CTP were added. 5P6 RNA polymerase (Ribopr) in the presence of RNAse inhibitor (RNasin; manufactured by Promega)
obeSP6 RNA Polymerase; Pr
(manufactured by omega) was added thereto, and the mixture was reacted at 37°C for 2 hours.

After the reaction, add DNA degrading enzyme DNASeI and heat at 37°C for 1 hour.
The reaction was allowed to proceed for 0 minutes. After protein removal with a chloroform-phenol mixture, the aqueous layer was applied to Sephadex G-50 gel, and 32P-labeled RNA was recovered and used as a probe.

Example 4 Separation of lymphocytes from southern hybridization human peripheral blood, chromosome D from lymphocytes
NA extraction and Southern hybridization were performed using the previously reported method of Drabkin et al.
H,A,,et at,,Proc,Natl
. ACad.

Sci, υS^82.464-468 (1985)).

Approximately 5 μQ of chromosomal DNA extracted from peripheral blood lymphocytes of healthy individuals and ALL patients was completely digested with the restriction enzymes listed separately in the table, and after 1.0% agarose gel electrophoresis,
The IOX S SC solution was used to transfer to a nitrocellulose filter. This filter is heated to 80℃
I baked it for about 2 hours. Using this nitrocellulose filter, probe B or probe
Southern hybridization was performed using C as a probe. After washing with 2XSSC solution at 50°C, autoradiography was performed.

As a result, when normal human genomic DNA was cut with the restriction enzyme Eco R■ and Southern hybridization was performed using probe B, one band of b at about 8.5 and one band of about 10
．． One 3Kb band, or about 8.5Kb and about 10.
Two 3Kb and three types of cases were observed.

The reason for such a difference in normal people is thought to be due to the presence of volmorphism in intron ■.

On the other hand, when performing the same hybridization using ALL patient; ALL-1 genomic DNA, approximately 8.5
Two bands, b at 9.7 and b at about 9.7, were observed.

This approximately 8.5 to b band is derived from the normal genome, but
The band at approximately 9.7 and b was shown to be an extra band due to chromosomal translocation. Also, ALL patients: ALL-
In the case of No. 2, two bands, b at about 8.5 and b at about 7, are seen, of which the band b at about 7 is an extra band related to chromosomal translocation. Also, this ALL-2
As a result of radiation therapy and other treatments after the onset of symptoms, these patients were completely cured. When we conducted a similar experiment using the genomic DNA of ALL-2 after complete recovery, only one band, b, was observed at approximately 8.5, indicating that the leukocytes in which the chromosome translocation had occurred had been completely removed. It shows that there is.

The above is explained using restriction enzyme ndI [I, Bam
HI.

Alternatively, the results were summarized in Table 2.

Also, using probe C, restriction enzyme EcoR■.

Table 3 summarizes the band patterns of Southern hyperdivisions performed on BCI+ and BCI+.

Table 2 Southern hybridization using probe B (unit: b) Table 3: Southern hybridization using probe C (unit: Kb) From the above results, further 5 of the restriction enzyme map shown in Figure 1 (2) was added. On the upstream side of the ' side, there is the first Bgt I cut point at about 1.9 Kb (about 0.8 Kb) counting from the EC0RI linker, and the first Bgt I cut point exists at about 2.4 B (about 1.4 Kb).
3 Kb) contains the first BamHI breakpoint, approximately 6 Kb).
．． 6b (approximately 5.5Kb) contains the first old ndI [l
It can be seen that there is a cutoff point (the numbers in parentheses are based on polmorphism). Therefore, gene fragments consisting of DNA or RNA molecules that hybridize to these parts can also be used for the purposes of the present invention.

[Brief explanation of the drawing]

Figure 1 (1) The entire restriction enzyme sequence exon of the BCR gene on the 22nd body is shown as a black bar, and the intron is shown as a thin line. E in the figure represents the restriction enzyme Eco RI. Figure 1 (2) Partial restriction of the first intron of the BCR gene The third E from the 3゛ side of the first intron in Figure 1 (1)
From the position 1 kb 5° upstream of the coRI cleavage site, the first Ec from the 3° side. A restriction enzyme map of DNA up to a position 1.5 Kb 3° downstream from the RI cleavage site is shown. The symbols in the figure indicate the following restriction enzymes. B:Bam HI, Bg:B(II
II, E: Eco RI, H: HindIII,
K; Kpn ■, X; Xho ■. Figure 1 (3) shows the position of the probe used in the probe experiment. Probe A is the first
0 upstream of the Xho I cleavage site on the 5° side of Figure (2)
．． 7 fragments of 15 Kb, probe B is shown in Figure 1 (2)
Probe C is a fragment of 0, 5 and b obtained by cutting the DNA fragment sandwiched between l)n I and Bam1-11 with the restriction enzyme Pvu I on the 5° side of the probe C, as shown in Figure 1 (2).
It is a 1°3 Kb fragment excised by the first Ba-) (I and the second Bam-II) from the 5° side of the phage clone encoding the human BCR gene according to the present invention. Fig. 1 shows the gene fragments of Fig. 1. Note that (2) to (4) in Fig. 1 correspond to the upper and lower parts, respectively.

Claims (1)

[Scope of Claims] 1. From a position 1 kb back to the 5' upstream side of the third EcoR I cleavage site from the 3' side in the first intron of the BCR gene on human chromosome 22, A gene fragment consisting of a nucleic acid molecule that hybridizes to at least a portion of the region up to the second HindIII cleavage site. 2. In the first intron of the BCR gene on human chromosome 22, from the position 1 kb 5' upstream from the third EcoR I cleavage site from the 3' side, to the second intron from the 3' side. A gene fragment consisting of an RNA molecule that hybridizes to at least a portion up to the HindIII cleavage site. 3. In the first intron of the BCR gene on human chromosome 22, from the position 1 kb 5' upstream from the third EcoR I cleavage site from the 3' side, to the second intron from the 3' side. DNA up to HindIII cleavage site
BamHI-BamHI, PvuII-Pv in the sequence
A gene fragment consisting of a nucleic acid molecule that hybridizes to a cleavage site 500 bases upstream from uII or XhoI-XhoI. 4. In the first intron of the BCR gene on human chromosome 22, from the position 1 kb 5' upstream from the third EcoR I cleavage site from the 3' side, to the second intron from the 3' side. DNA up to HindIII cleavage site
BamHI-BamHI, PvuII-Pvu in the sequence
II, or a gene fragment consisting of an RNA molecule that hybridizes to a cleavage site 500 bases upstream from Xho I - Xho I. 5. The above hybridization is carried out with the above BamH I
-Ba-H I , PvuII-PvuII, or Xho I -
5. The gene fragment according to claim 3 or 4, which corresponds substantially completely to the DNA sequence of the cleavage site 500 bases upstream from Xho I. 6. A probe consisting of the gene fragment according to any one of claims 1 to 5, which is labeled with a detectable substance. 7. A method for detecting chromosomal abnormality, which comprises cleaving genomic DNA of a subject with a restriction enzyme, performing Southern hybridization using the probe according to claim 6, and then detecting a band.